Plant growth promoting rhizobacteria improve growth and yield related attributes of chili under low nitrogen availability

Nitrogen (N) is a macronutrient desired by crop plants in large quantities. However, hiking fertilizer prices need alternative N sources for reducing its requirements through appropriate management practices. Plant growth promoting rhizobacteria (PGPR) are well-known for their role in lowering N requirements of crop plants. This study assessed the impact of PGPR inoculation on growth, allometry and biochemical traits of chili under different N doses. Two PGPR, i.e., Azospirillum ‘Er-20’ (nitrogen fixing) and Agrobacterium ‘Ca-18’ (phosphorous solubilizing) were used for inoculation, while control treatment had no PGPR inoculation. Six N doses, i.e., 100, 80, 75, 70, 60 and 50% of the N required by chili were included in the study. Data relating to growth traits, biochemical attributes and yield related traits were recorded. Interaction among N doses and PGPR inoculation significantly altered all growth traits, biochemical attributes and yield related traits. The highest values of the recorded traits were observed for 100% N with and without PGPR inoculation and 75% N with PGPR inoculation. The lowest values of the recorded traits were noted for 50% N without PGPR inoculation. The PGPR inoculation improved the measured traits compared to the traits recorded noted in same N dose without PGPR inoculation. Results revealed that PGPR had the potential to lower 25% N requirement for chili. Therefore, it is recommended that PGPR must be used in chili cultivation to lower N requirements


Introduction
Nitrogen (N), phosphorous (P) and potassium (K) are the important mineral nutrients for the optimum growth and production of plants in commercial agricultural systems [1]. Nitrogen a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 availability is the major hurdle in chili production and higher N uptake could decrease the quality. Therefore, eco-friendly management options are needed to lower the N requirement of the crop for higher productivity. As described above, PGPRs have the potential to improve crop productivity under sub-optimal conditions. However, these have been less explored for improving chili production under low N availability.
Therefore, this study assessed the role of synergistic inoculation of N-fixing (Azospirillum Er-20) and P-solubilizing (Agrobacterium Ca-18) PGPRs for improving chili production under low N availability. Similarly, different N doses were used to assess the yield losses caused by decreasing N availability. It was hypothesized that different N doses will differ in growth and productivity of chili. It was further hypothesized that PGPRs' inoculation will improve growth and yield of chili under reducing N doses.

Plant materials, PGPRs' source, and seedling inoculation
Chili seedlings were procured from Jafar group, Multan, Pakistan (30.12908˚N, 71.37459˚E). Two PGPR strains, i.e., Azospirillum Er-20 (N-fixing) and Agrobacterium Ca-18 (P-solubilizing) were obtained from National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan (31.39819˚N, 73.02575˚E). The bacterial strains were grown in 1 L LB broth medium at 28˚C for 48 hours to get the optimum growth (CFU �10 9 mL -1 ). Bacterial cells were then harvested by centrifugation at 4000 × g for 20 min at 4˚C and suspended in 1 L of saline solution (w/v 0.89% NaCl).
The PGPR mixture was prepared by adding 5 g PGPR and 100 ml water. Before transplanting, the roots of seedlings were dipped in PGPR mixture for half hour. Transplanting was completed in the morning and irrigation was applied soon after transplanting.

Treatments and experimental design
The seedlings were either dipped (PGPR inoculation) or not dipped (no PGPR inoculation) in PGPR mixture. The P and K were applied at their recommended doses, whereas N doses were 100, 80, 75, 60, 60 and 50% of the recommended N for chili. Seedlings were transplanted on 60 cm apart ridges 60 cm by keeping plant to plant distance of 30 cm. Seedlings were transplanted on Dec 22, 2015. Irrigation was applied just after transplanting the seedlings and 2 nd irrigation was applied after 2 weeks of transplanting. Further irrigation was applied according to the field and weather condition. All other cultural practices were followed as recommended for growing chili crop under field conditions. Insects, pests were controlled by the foliar application of insecticide Actara (0.63 g/l). collected from each treatment. Data relating to total number of fruits per plant, fresh and dry weight of fruit, fruit length and fruit width were also recorded at harvest. Ten plants were randomly selected from each experimental unit. All fruits on the plants were harvested during each picking and weighed. From the fresh weight, yield/ha was calculated. Leaf samples were collected for determining antioxidant capacity and total phenolic following Singleton and Rossi [33]. Samples were prepared by adding 29% water, 1% acetic acid, and 70% ethanol. One gram sample was mixed with 10 ml of the prepared solution. The mixture was clarified and then stored at -80˚C for further analysis. Antioxidant capacity was measured by the method of Brand-Williams et al. [34]. About 30 μL sample was measured from the extracted samples and mixed in 2.97 mL of 0.1% DPPH solutions by using spectrophotometer at the 515 nm the antioxidant action was expressed. The antioxidant action was estimated by following the antioxidant capacity excepting change in formula for activity.
The carotenoid content of fruit was estimated by the method of Kichtenthaler and Wellburn [36]. One gram of fresh green chili was mixed with 80% acetone and volume was raised to10 ml. Samples were centrifuged at 800 rpm for 5 minutes and read at 470 nanometers. Carotenoids were computed by the formula given below.
Carotenoids content mg=g fresh weight ¼ 1000ðA470Þ À 3:27 ðChlÀ aÞ À 104ðChl bÞ=227 The total phenolic content was analyzed by following Singleton and Rossi [33]. Folin-Ciocalteu's phenols mixture, extracted sample and distilled water were mixed in ratio of 1:1:20 (v/ v) respectively. The subsequent solution was kept in dark for 8 minutes. Later, 10 ml of 7% (w/ v) sodium carbonate was added into it and absorbance was taken after 2 hours at 750 nm by using spectrophotometer. The total phenolic contents were stated in microgram gallic acid equivalent gram-1 fresh weight basis (GAE/g fw). The chlorophyll content of leaves was taken with the help of chlorophyll meter. From each replication five plants were randomly selected for chlorophyll determination.

Statistical analysis
Data collected were analyzed statistically by using Fisher Analysis of variance (ANOVA) technique [37]. Least significant difference (LSD) test at 5% level of probability were applied for the separation of treatment means. Statistix 8.1 analytical software (Tallahassee Florida, USA) was used for this purpose.

Results
Individual and interactive effects of PGPR inoculation and N doses significantly altered different growth traits, including plant height at flowering and maturity, stem diameter, root length, and fresh and dry weights of roots and shoot with some exceptions for individual effects of PGPR inoculation for stem diameter, root length and shoot fresh weight (Table 1).
Different growth traits, including plant height at flowering and maturity, stem diameter, root length, and fresh and dry weights of roots and shoot were significantly affected by interactive effect of PGRP inoculation and N doses ( Table 2). Overall decreasing N availability reduced all measured growth traits under both PGPR inoculation and no inoculation. However, this decrease was more severe in no PGPR inoculation compared with PGPR inoculation ( Table 2). The highest values of growth traits were observed for the interaction of PGPR inoculation with 100 and 75% N availability and no PGPR inoculation with 100% N availability. However, the lowest values of growth traits were recorded for no PGPR inoculation interaction with 50% N availability ( Table 2). Individual and interactive effects of PGPR inoculation and N doses significantly altered different reproductive and yield-related traits, including yield, chlorophyll contents, total number of fruits, fruit fresh and dry weight and length and width (Table 3).
Different reproductive and yield-related traits, including yield, chlorophyll contents, total number of fruits, fruit fresh and dry weight and length and width were significantly affected by interactive effect of PGRP inoculation and N doses (Table 4). Overall decreasing N availability reduced all measured reproductive and yield-related traits under both PGPR inoculation and no inoculation. However, this decrease was more severe in no PGPR inoculation compared with PGPR inoculation ( Table 4). The highest values of reproductive and yield-related traits were observed for the interaction of PGPR inoculation with 100 and 75% N availability and no PGPR inoculation with 100% N availability. However, the lowest values of growth traits were recorded for no PGPR inoculation interaction with 50% N availability (Table 4).
Individual and interactive effects of PGPR inoculation and N doses significantly altered different biochemical traits, including antioxidant activity and capacity, total phenolic contents, carotenoids, leaf chlorophyll content and fruit chlorophyll a and b contents with some exceptions (Table 5). However, individual effects of PGPR inoculation were non-significant for antioxidant activity and capacity and chlorophyll a in the fruit (Table 5).
Different biochemical traits, including antioxidant activity and capacity, total phenolic contents, carotenoids, leaf chlorophyll content and fruit chlorophyll a and b contents were significantly affected by interactive effect of PGRP inoculation and N doses ( Table 6). Overall decreasing N availability reduced all measured biochemical traits under both PGPR inoculation and no inoculation. However, this decrease was more severe in no PGPR inoculation compared with PGPR inoculation ( Table 6). The highest values of biochemical traits were observed for the interaction of PGPR inoculation with 100 and 75% N availability and no Here, B 1 = no bacteria inoculation, B 2 = bacteria inoculation, F 1 = 50% N, F 2 = 60% N, F 3 = 70% N, F 4 = 75% N, F 5 = 80% N, F 6 = 100% N, Means followed by different letters within a column statistically differ from each other (p>0.05).
PGPR inoculation with 100% N availability. However, the lowest values of growth traits were recorded for no PGPR inoculation interaction with 50% N availability (Table 6).

Discussion
Different growth, yield, and biochemical attributes of chili significantly differed among different N doses and confirmed our first hypothesis. Likewise, PGPR inoculation significantly increased growth, yield, and biochemical attributes of chili compared to no PGPR inoculation confirming our second hypothesis. Overall, PGPR used in the current study have potential to reduce N requirement of chili by 25% without any yield losses. Similar results for PGPR inoculation have been reported for different crops like cucumber, tomato and legumes [11,12,[38][39][40]. However, these studies used only one PGPR, while we used two PGPRs indicating that both worked synergistically to improve the studied traits of chili. The PGPRs probably improved these traits by producing different compounds (like phytohormones, organic acids and siderophore), N-fixing, P solubilization and production of biologically active constituents [16][17][18][19][20].

PLOS ONE
Plant growth promoting rhizobacteria lower nitrogen requirements of chili u It has been reported three PGPR isolates, i.e., B. stratosphericus-NFB3, B. cereus MNB1 and P. simie-NTB2 increased plant of chili over the untreated plants [41]. Bi et al. [38] reported that plant height was improved by the application of manure and PGPR in cucumber and tomato. Similarly, PGPR inoculation increased the fresh root weight of cauliflower [42].
A significant increase in the fresh weight of root with PGPR inoculation in cabbage seeds was reported by Turan et al. [42]. Hence, the results of the present study are in the agreement with the findings of previous workers. Similarly, Kanchana et al. [43] reported that fruit fresh and dry weight increased in chili var K1.) due to the interaction effect of PGPR. Likewise, Pirlak et al. [44] reported that foliar application of PGPR significantly increased fruit fresh weight (4.2-7.5%) in "Starkrimson" and fruit fresh weight (6.5-8.7%) in "Granny Smith".
Foliar and floral applications of different PGPR strains, i.e., Bacillus mycoides T8 and Bacillus subtilis OSU-142 alone or in combinations significantly increased the fruit length in quince [45]. Hence, the results of the present study are in the agreement with earlier findings. Similarly, PGPR are known to increase the yield of chickpea and other important crops when the seeds of these crops are inoculated with PGPRs [11,12].

Conclusion
The current study indicated that decreasing N availability suppressed growth, yield, and biochemical attributes of chili. However, PGPR inoculation significantly improved these traits even under low N availability. Overall, the PGPR inoculation with 75% N availability produced similar traits as of 100% N availability. Thus, it is concluded that PGPR has the potential to lower N requirement of chills crop; thus, these can be used to improve chili productivity with low N availability.